This invention is related to the Molecular Pharmacology field and especially to the development of peptides useful for treating HPV-associated epithelial tumors as it permits the blocking of the Casein Kinase II (CKII) phosphorylation domain by direct interaction with such a site.
The CKII is a threonine/Serine enzyme involved in the cellular proliferation and its intracellular localization is mainly into nucleus during malignant transformation process (Tawfic S, Yu S, Wang H, Faust R, Davis A, Ahmed K, 2001, Histol. Histopathol. 16:573-582).
Based on the findings reporting CKII high levels in different epithelial solid tumors, it has been assumed that phosphorylation elicited by this enzyme is an important event in malignant transformation and a tumor progression marker (Seldin D C, Leder P, 1995, Science 267:894-897) (Faust R A, Gapany M, Tristani P, Davis A, Adams G L, Ahmed K, 1996, Cancer Letters 101:31-35). On the other hand, the CKII over expression in transgenic mice leads to the tumorigenesis in the mammary glands by increasing the Wnt/beta-catenine signal transduction pathways on these mammary epithelial cells (Landesman-Bollag E, Romien-Mourez R, Song D H, Sonenshein G E, Cardiff R D, Seldin D C, 2001, Oncogene 20:3247-3257).
Among epithelial tumors, those originated by HPVs represent a great fraction. For instant, most of the genitourinary tumors are associated to these oncoviruses and the presence of HPV DNA sequences has been demonstrated in 99.7% of the tumors coming from squamous cervical cells (Walboormers J M, Jacobs M V, Manos M M, Bosch F X, Kummer J A, Shah K V, Snijders P J, Peto J, Meijer C J, Muñoz N, 1999, J. Pathol 189:12-19). Likewise, the WHO has reported about 500 000 cervical cancer patients annually worldwide (Parkin D M, Laara E, Muir C S, 1980, Int. J. Cancer 41:184-1972). In Cuba, 370 women with cervical cancer die annually due to this disease (Organizacion Panamericana de la Salud, 1999, Basic Country Health Profiles for the Americas. Cuba, 206-219)
HPVs are classified in oncogenic and not oncogenic according to whether the lesions progress toward malignancy or not (Lorincz A T, Temple G F, Kurman R J, Jenson A B, Lancaster W D, 1987, J. Natl. Cancer Inst. 79:671-677). HPV-16 and -18 are associated to intraepithelial neoplasia that generally progress toward malignancy and also both HPV types are associated to more than 90% of the dysplasias and cervical carcinomas (Fujinaga Y, Shimada M, Okasawa K, Fukushima M, Kato I, Fujinaga K, 1991 J. Gen. Virol 72:1039-1044).
As no therapeutic or prophylactic vaccine is still available for eradication of HPV-associated tumors, the employment of inhibitors targeting viral transcription and viral oncoproteins, become more attractive. Biomodulators like IFNs have been used with some efficacy in certain HPV-associated diseases like condyloma, plantar warts, and respiratory papillomatosis (Koromilas A E, Li S, Matlashewski G, 2001. Cytokine & Growth Factor Reviews 12:157-170). In previous experiments on HPV-transformed cells (HeLa), we have demonstrated that continuous exposition with IFN alpha produces a reversion of the malignant phenotype of these cells with the concomitant inhibition of the HPV mRNA expression (López-Ocejo O, Perea S E, Reyes A, Vigoa L, López-Saura P, 1993. J. IFN Res 13:369-375). In the same cellular model, we found that IFN alpha modulates the HPV mRNA through the repression of endogenous viral transcription (Perea S E, López-Ocejo O, García-Milián R, Araña M J, 1995, J. IFN & Cytokine Res 15:495-501). In agreement with the results obtained in cell lines, we observed that IFN alpha treatment modulated the mRNA expression in a pilot study in cervical cancer patients (García-Milián R, Rios M A, Díaz D, Silveira M, Guilar O, Amigó M, Araña M J, Perea S E, 1996, J. IFN and Cytokine Res 16:709-713). In spite of the promissory findings about the use of IFN as regulator of the HPV mRNA expression, mounting data indicate a variable IFN response and the resistance phenomenon toward this cytokine has been reported between the 40 and 50% of the patients during clinical trials (Arany I, Tyring S K, Stanley M A, Tomai M A, Miller R L, Smith M H, McDermott, D J, Slade H B, 1999, Antiviral Res 43:55-63). Some molecular and clinical evidences indicate that E7 oncoprotein plays a central role on the IFN-resistance phenomenon. For example, it has been reported that E7 binds to the IFN-induced transcription factor (p48) thus affecting the IFN response by blocking the transcriptional activation (Barnard P and McMillan N A J, 1999, Virology 259:305-313). Furthermore, the alteration of the IFN regulatory factor (IRF-1) in the presence of E7 has been also reported (Park J S, Kim E J, Kwon H J, Hwang E S, Namkoong S E, Um S J, 2000, J Biol Chem 275:6764-6769) (Perea S E, Massimi P, Banks L, 2000, J Mol Med 5:661-666). In clinical trials, the IFN response has been regarded to be depending on the E7 expression in the HPV-containing lesions (Frazer I H, McMillan N A J, 1997, Papillomatosis and condyloma acuminate. Clinical Applications of the Interferons (R Stuart Harris and R D Penny, eds) Pp 79-90. Chapman and Hall Medical, London). The E7 oncoprotein plays an essential role on the malignant transformation elicited by these oncogenic viruses. Thus, it has been demonstrated that E7-induced immortalization of primary cells leads to mutations and chromosomal aberrations since the beginning of the immortalization process (Mougin C, Humbey O, Gay C, Riethmuller D, 2000, J. Gynecol Obstet. Biol. Reprod 29:13-20). On the other hand, we have demonstrated that stable transfection with the E7 gene leads to the development of a IFN-resistant phenotype on sensitive tumor cells (Moro A, Calixto A, Suárez E, Araña M J, Perea S E, 1998, Bioch Bioph Res Comm 245:752-756). Likewise, it has been reported that E7 oncoprotein binds and blocks the function of tumor suppressor genes like the Retinoblastoma (Rb) and the Insulin-like Growth Factor Binding Protein-3″ (IGFBP-3) through the Cys 24 and the C-terminal domain respectively (Nevins J R, 1992, Science 258:424-429) (Zwerschke W and Jansen-Durr P, 2000, Advances in Cancer Res 78:1-29). Similarly, the Ser 31/Ser 32 doublets in E7 protein have shown to be substrate for the CKII enzyme (Hashida T, Yasumoto S, 1990, Biochem. Biophys Res. Comm 172:958-964) and this domain is essential for both the transformant capacity of this oncoprotein (Barbosa M S, Edmonds C, Fisher C, Schiller J T, Lowy D R, Vousden K, 1990, EMBO J 9:153-160) (Chien W-M, Parker J N, Schmidt-Grimminger D-C, Broker T R, Chow L T, 2000, Cell Growth & Differentiation 11:425-435) and the inhibition of the IFN signaling cascade (Perea S E, López-Ocejo O, García Milián R, Banks L, Araña M J, 1996, Eur. Cytokine Net 7:503).
Based on the role of the CKII phosphorylation site in the HPV-resistance to IFN and cancer development, the designing of drugs blocking such a domain could become as useful tools for cancer therapy. Molecules inhibiting the CKII phosphorylation site either on E7 or in other cell substrates have not been described so far.
Concerning the E7 oncoprotein, only peptides blocking the Rb binding site (Cys 24) (Webster K R, Koleman K G, 1997, U.S. Pat. No. 5,625,031) (Oliff A I, Riemen M W, EP 0412762 A2 910213) and other C-terminal regions (39-98) have been described (Pidder J-D, Zwerschke W, 2000, EP0969013).
Some vaccine candidates focused to develop HPV E7-specific CTL response have been so far described in clinical or pre-clinical trials (Chen C, Wang C C, Hung C, Pardoll D M, Wu T, 2000, Vaccine 18:2015-2022) (Chen C H, Ji H, Suh K W, Choti M A, Pardoll D M, Wu T C, 1999, Gene Ther 12:1972-1981). However, the approaches focused to the CTL response face different obstacles related to the HPV biology. For instant, HPV oncogenic types down-regulate the MHC class I antigens which are essential for the CTL response (Connor M E, Stern P L, 1990, Int J Cancer 46:1029-1034). Furthermore, E7 expression has been associated with local immunosuppression at the tumor environment and this could also affect the appropriated development of the CTL response (Le Buanec H, D'Anna R, Lachgar A, Zagury J F, Bernard J, Ittlele D, d'Alessio P, Hallez S, Giannouli C, Burny A, Bizzini B, Gallo R C, Zagury D, 1999, Biomed Pharmacother 53:424-431) (Lee S J, Cho Y S, Shim J H, Lee K A, Ko K K, Choe Y K, Park S N, Hoshino T, Kim S, Dinarello C A, Yoon D Y, 2001, J Immunol 167:497-504). According to the above elements, it seems that combining CTL vaccines and pharmaceuticals targeting E7, could be of great perspectives.
Likewise, the approach of preventive HPV-vaccines faces a high benefit and cost risk due to different biological and social aspects including: 1) Long latency period after the HPV primary infection, 2) poor understanding of the HPV infection mechanism, 3) no animal model for the appropriated HPV propagation, 4) specie specificity and 5) the evaluation of the social impact of a preventive HPV vaccine could take quite long. Therefore, the using of pharmaceuticals specifically targeting viral oncoproteins could provide advantages over those approaches focused to the manipulation of the immune system.